The present invention relates in general to anticancer agents, and more specifically to synthetic analogs of deoxypreussomerin, palmarumycin CP1 and related naphthoquinone spiroketals, which exhibit antimitotic activity.
The cell cycle consists of series of stages abbreviated G1- S - G2 - M. G1 stands for gap number 1; S for synthesis (DNA synthesis occurs); G2 for gap number 2 and M for mitosis or cell division. Control of cell division is very complex and involves regulation at a number of levels. In cancerous cells, the normal regulatory processes are somehow disrupted and cell growth is uncontrolled.
Tubulin is a protein that polymerizes into long chains or filaments that form microtubules. Microtubules are hollow fibers, which serve as kind of a skeletal system for living cells. Microtubules have the ability to shift through various formations, thus allowing the cell to undergo mitosis. The formation-shifting of microtubules is made possible by the flexibility of tubulin monomers, especially in the presence of agents/drugs and proteins that bind tubulin.
Interest in tubulin has increased recently because a natural substance (paclitaxel) found in the bark of the Pacific yew tree, was shown in clinical tests to be an effective treatment for a number of cancers including ovarian, breast, and lung. Paclitaxel prevents cell division by promoting the assembly of and inhibiting the disassembly of microtubules.
Cell cycle checkpoints are critical regulators of genome integrity and faithful cell replication. One of the main abnormalities in human tumors cells is the loss of the G1 phase checkpoint, which not only permits cellular replication but also encourages genomic instability. Consequently, enforcement of the G2/M checkpoint represents an attractive mode of action for new antineoplastic agents. G2/M progression is tightly regulated by several cellular macromolecules, including tubulins, and microtubule-associated proteins and motor proteins, such as kinesins and dynesins. An additional essential regulator is the maturation/M-phase promoting factor comprising Cdk1/cyclin B. Cdk1/cyclin B itself is regulated by a complex group of positive and negative regulating kinases. In mammalian cells, these include weel, mytt, cyclin activating kinase, Chk1 and cds1 kinases. In addition, Cdc25 phosphatases, which are also regulated by other kinases and phosphatases, are responsible for the activation of Cdk1.
Inhibitors of tubulin polymerization or depolymerization are widely available but only a few disrupters of other regulators of G2/M progression have been identified. For example, several small molecule inhibitors of Cdc25 that block G2/M progression have been identified, but those compounds also affect G1 transition (Tamura K, Rice RL, Wipf P and Lazo JS (1999) Dual G1 and G2/M phase inhibition by SC-xcex1xcex1xcex49, a combinatorially derived Cdc25 phosphatase inhibitor. Oncogene 18:6989-6996, Tamura K, Southwick EC, Kerns J, Rosi K, Carr BI, Wilcox C and Lazo JS (2000) Cdc25 inhibition and cell cycle arrest by a synthetic thioalkyl vitamin K analogue. Cancer Res 60:1317-1325). Others have recently isolated a novel mitotic blocker that appears to act as a specific inhibitor of a mitotic kinesin (Mayer TU, Kapoor TM, Haggarty SJ, King RW, Schreiber SL and Mitchison TJ (1999) Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen. Science 286:971-974). Nonetheless, there continues to be a great need for pharmacologically distinct agents both to investigate the G2/M progression process and as new pharmacophores for drug design strategies.
The antifungal metabolites, preussomerins A-F, were identified in 1990 by Gloer and coworkers during the course of an investigation of chemical agents involved in interspecies competition among coprophilous (dung-colonizing) fungi. (Weber, H. A.; Baenziger, N. C.; Gloer, J. B. J Am. Chem. Soc. 1990, 112, 6718). In addition to those early reports from Preussia isomera Cain samples, preussomerins were later also discovered in the endophytic fungus Harmonerna dematioides (Polish J. D.; Dombrowski, A. W.; Tsou, N. N.; Salituro, G. M.; Curotto, J. E. Mycologia 1993, 85, 62). Other reports of an epoxy naphthalenediol spiroketal compound, bipendensin, have been published (Connolly, J. D. 4th International Symposium and Pakistan-US. Binational Workshop on Natural Products Chemistry, Karachi, Pakistan, January 1990 and Connolly, J. D.: Structural elucidation of some natural products. In Studies in Natural Products Chemistry, Vol 9. Ed., Atta-ur-Rahman, pp. 256-258, Elsevier Science Publishers B. V., Amsterdam, 1991). Bipendensin was isolated in very small amounts from wood samples of the African tree Afzelia bipendensis. A compound having the same gross structure as bipendensin was isolated in 1994 from an unidentified Coniothyium fungus collected from forest soil on West Borneo, and was named palmarumycin C11, (Krohn, K.; Michel, A.; Florke, U.; Aust, H.-J.; Draeger, S.; Schulz, B. Liebigs Ann. Chem. 1994, 1099).
The pentacyclic palmarumycins are structurally unique natural products with both antifungal and antibacterial activities, but their antineoplastic effects are not well established. The naphthoquinone acetals, palmarumycins, diepoxins and deoxypreussomerins are structurally unique fungal metabolites with both antifungal and antibacterial activities, but their antiproliferative activity against malignant mammalian cells has not been extensively studied. (Wipf, P and June JK (1998) Total synthesis of palmarumycin CP1 and (xc2x1)-deoxypreussomerin A. J Org Chem 63:3530-3531: Schlingmann GRR, West LP, Milne CJ and Carter GT (1993) Diepoxins, novel fungal metabolites with antibiotic activity. Tetrahedron Lett 34:7225-7228: Krohn K, Michel A, Florke U, Aust H-J, Draeger S and Schulz B (1994) Palmarumycins C1-C6 from Coniothyrium: Isolation, structure elucidation, and biological activity. Liebigs Ann Chem 1994:1099-1108). Biological studies have been limited due to the extraordinary synthetic challenges associated with the extensive levels of oxygenation and the highly electrophilic functionality present in these spiroketal natural products.
Since its discovery in the early 1960s, the thioredoxinxe2x80x94thioredoxin reductase system has been the subject of intense pharmacological studies (Williams,C.H. Eur.JBiochem. 2000, 267, 6101). The two redox active proteins have been isolated from many species, and their medical interest is based in part on their value as indicators of widespread diseases such as rheumatoid arthritis, AIDS, and cancer. The cytosolic 12 kDa thioredoxin-1 (Trx-1) is the major cellular protein disulfide reductase and its dithiol-disulfide active site cysteine pair (CXXC) serves as electron donor for enzymes such as ribonucleotide reductase, methionine sulfoxide reductase, and transcription factors including NF-xcexaB and the Ref-1-dependent AP-1 (Arn r,E.S.J.; Holmgren,A. Eur.JBiochem. 2000, 267, 6102). Therefore, thioredoxin-l is critical for cellular redox regulation, signaling, and regulation of protein function as well as defense against oxidative stress and control of growth and apoptosis. (Davis,W.; Ronai,Z.; Tew,K.D. JPharm.Exp. Ther. 2001, 296, 1). Thioredoxin-1 acts in concert with the glutathionexe2x80x94glutathione reductase system but with a rate of reaction orders of magnitude faster,and lack of cytosolic mammalian thioredoxin is embryonically lethal. Eukaryotic thioredoxin reductases (TrxR) are 112-130 kDa, selenium-dependent dimeric flavoproteins that also reduce substrates such as hydroperoxides or vitamin C (Williams, C.H.; Arscott, L.D.; Miller,S.; Lennon,B.W.; Ludwig,M.L.; Wang,P.-F.; Veine,D.M.; Becker,K.; Schirmer,R.H. Eur.JBiochem. 2000, 267,6110). These reductases contain redox-active selenylsulfide-selenolthiol active sites and are inhibited by aurothioglucose and auranofin (K; 4 nM). (Becker,K.; Gromer,S.; Schirmer,R.H.; Mller,S. Eur.JBiochem.2000, 267, 6118). NADPH serves as reducing agent of Trx-1 via TrxR.
Pathophysiological effects of Trx-1/TrxR are indicated by Trx-1 overexpression in human tumors such as lung, colorectal and cervical cancers and leukemia, and secreted Trx-1 stimulates cancer cell growth and decreases sensitivity to induced apoptosis (Powis,G.; Kirkpatrick,D.L.; Angulo,M.; Baker,A. Chem.-Biol.Interactions 1998, 111 ,23). The Trx-1/TrxR system is therefore an important target for chemotherapeutic intervention. Alkyl 2-imidazolyl disulfides were found to be inhibitors of Trx-1/TrxR with IC50""s of 31/37 xcexcM, respectively; these disulfides block MCF-7 human breast cancer cells in the G2/M phase of the cell cycle and suppress the growth of several human primary tumors in the NCI 60 cancer cell line panel (Vogt,A.; Tamura,K.; Watson,S.; Lazo,J. S. J. Pharm.Exp.Ther. 2000, 294 ,1070). A COMPARE analysis revealed the most potent Trx-1/TrxR inhibitor known to date, the para-quinone NSC401005 which is the natural product pleurotin (Kunkel,M.W.; Kirkpatrick,D.L.; Johnson,J.I.; Powis,G. Anti-Cancer Drug Des. 1997, 12, 659). The IC 50 of NSC401005 was determined as 0.17 xcexcM; however, the average GI 50 of this compound in the 60 cell line panel was only 21.5 xcexcM. Although inhibitors of TrxR such as auranofin and nitrosoureas are quite effective, the search for new, more specific, and less toxic compounds is well justified.
Therefore, a need exists in the art for new chemical compounds that block G2/M phase transition. Such compounds would find use as pharmacological probes and possible lead structures for therapeutic agents. These compounds may include inhibitors of the thioredoxinxe2x80x94thioredoxin reductase system which are less toxic than current compounds.
The inventors have developed an efficient synthetic approach toward palmarumycins, diepoxins and deoxypreussomerins and have generated a library of analogs. A number of these analogs inhibit the thioredoxinxe2x80x94thioredoxin reductase system. The inventors have examined the antiproliferative actions of pentacyclic palmarumycins against tumor cells using a temperature sensitive tsFT210 mouse mammary carcinoma cell line and found that a new palmarumycin analog, [8-(furan-3-ylmethoxy)-1-oxo- 1,4-dihydronaphthalene-4-spiro-2 xe2x80x2-naphtho[ 1xe2x80x3,8xe2x80x3-de] [1xe2x80x2,3xe2x80x2] [dioxin] herein termed SR-7, prominently blocked mammalian cell cycle transition in G2/M but not in G1 phase. The inventors found no evidence for inhibition of the critical mitosis-controlling cyclin-dependent kinase, Cdk1, or its regulator, the dual specificity phosphatase Cdc25. Moreover, Cdk1 was hypophosphorylated and not directly inhibited by SR-7. SR-7 also failed in vitro to hypernucleate bovine tubulin, did not compete with colchicine for tubulin binding, and only modestly blocked GTP-induced assembly. In addition, SR-7 caused almost equal inhibition of paclitaxel-sensitive and -resistant cell growth. Moreover, unlike benchmark tubulin disrupting agents, SR-7 did not cause hyperphosphorylation of the antiapoptotic protein Bcl-2. Thus, SR-7 can inhibit G2/M transition by a mechanism that appears to be independent of marked tubulin disruption.
These and other advantages and benefits of the present invention will be apparent from the Detailed Description of the Invention herein below.